DO DNA DISTANCES REVEAL AVIAN PHYLOGENY?

L. J. GibsonGeoscience Research Institute

Evolutionary classification methods attempt to group species according to
how closely they are related. But how are relationships measured? A review of the
literature pertinent to DNA distances indicates that independent approaches may give
conflicting results.

INTRODUCTION

Two goals of evolutionary studies are to determine the relationships
among organisms and the mechanisms by which change occurs (Sibley and Ahlquist 1986). For
various reasons, both goals have been elusive. Relationships among species are often
difficult to establish, especially because different methods of comparison may give
different results. Such conflicting results are attributed to convergence, parallelism,
reversals, and unequal rates of change. These problems are usually explained as the result
of natural selection. By this reasoning, the best method for determining phylogeny would
be to use some feature not controlled by selection.
If, as has been suggested (Kimura 1968), most point mutations are
neutral to selection, such mutations could provide a record of ancestry. It should be
possible to identify mutations and determine relationships by comparing DNA sequences.
Several methods of estimating DNA differences have been devised. Comparison of amino-acid
sequences gives an estimate of the corresponding differences in DNA sequence for
relatively small amounts of DNA. Actual sequencing of entire genomes is not yet practical.
Restriction framing mapping may be the most efficient molecular method of phylogenetic
estimation at the present time. Discussion of these methods is beyond the scope of this
article. DNA/DNA hybridization is a very crude method of estimating sequence differences,
but is the subject of this paper because of the large amount of published data for birds,
and especially the single order of perching birds. In this paper, the term DNA distance is
used only to refer to results of the DNA/DNA hybridization technique.Neutral mutation theory. Based on calculations of the
rate of mutations thought to be needed to explain amino-acid differences in proteins from
different species, Kimura (1968) proposed that most point mutations must be nearly neutral
to selection in order to explain their apparently rapid rate of fixation (see also Kimura
1979). The suggestion that mutations might be neutral led to the development of a
theoretical explanation for their neutrality.
Mutations may be neutral either because they occur in DNA that is
non-functional or because the mutation does not alter the function of the sequence. In
many organisms from 20% to 50% of the genome may consist of highly repetitive DNA (Britten
and Davidson 1971), most of which has no known function. In addition, it appears that most
of the single-copy DNA is present as intervening sequences (introns) that are not
translated (e.g., see Wozney et al. 1981). Most mutations in multiple-copy DNA or in
introns should theoretically have no effect on the organism (but see below).
Mutations may also be neutral because they do not change the function
of a translated DNA sequence. Within coding sequences, many mutations in the third base of
a codon do not alter the meaning of the codon (Jukes 1980). Such "silent
substitutions" could account for as much as 17% difference between two functionally
identical DNA sequences (McCarthy and Farquhar 1972). Also, mutations that result in
substitution of an amino acid for a very similar amino acid might have no noticeable
effect on the phenotype, and be essentially neutral.
The proposal that most mutations are neutral seems to have been
generally accepted, although not universally (e.g., Bernardi and Bernardi 1986, Gillespie
1986). However, recent evidence shows that an active gene may be contained within an
intron of another gene (Henikoff et al. 1986), and that two genes may overlap each other
on opposite DNA strands (Adelman et al. 1987). Nesting and overlapping of genes are
believed to be uncommon in vertebrates, but mutations in such DNA would probably not be
neutral. It should be noted that the theory stressing the importance of neutral mutations
in evolution was originally proposed (Kimura 1968) to explain the larger-than-expected
differences in amino-acid sequences among several species of mammals assumed to have a
common ancestry datable from the fossil record. If one accepts the possibility of
separately created lineages the problem of explaining large differences between species
disappears, and the issue of neutral mutations becomes less important.DNA clock and systematics. There are large numbers of
genes in the genome of a multicellular organism, and it has been argued that, even if
mutation rates vary for different genes, the average rate of nucleotide substitution for
all genes would be uniform over long periods of time (Sibley and Ahlquist 1983a, 1986). If
true, the difference in DNA sequences between two species would be a function of the time
since their divergence. This concept forms the basis of the purported "DNA
clock".
Sibley and Ahlquist (1983a) assert that DNA/DNA hybridization results
give an accurate estimate of the overall sequence difference between any two species, and
(1983a, 1986) that the resulting measurements of DNA distance provide a tool for
accurately determining relationships and estimating times of origin of the species.
Sibley, Ahlquist and Sheldon (1987) have suggested that DNA comparisons are more reliable
than morphological comparisons in determining phylogeny because DNA sequences are not
subject to convergence.
Recently it has been shown that differences in DNA sequences are not
necessarily related to the supposed age of lineages (Sheldon 1987b, Catzeflis et al.
1987), thus invalidating the use of DNA/DNA hybridization distance data as a clock.
However, the data show some interesting patterns and further investigation seems
worthwhile.

DESCRIPTION OF TECHNIQUES

DNA preparation. The following description of the
technique is based on Sibley and Ahlquist (1983a, 1986). DNA is collected from red blood
cells or other appropriate tissue. After purification, the DNA is sheared by sonication
into fragments that average about 500 base pairs in length. These fragments are boiled to
separate the strands, then the mixture is partially cooled. Since the number of copies of
repetitive sequences in the mixture is much greater than those of single-copy DNA, they
will reassociate faster. When the mixture is passed over a hydroxyapatite column,
double-stranded reassociated fragments bind to the column, while the single-stranded
fragments of single-copy DNA are collected in the effluent. This single-copy DNA is
believed to contain 95-98% of the different sequences present (Sibley and Ahlquist 1983a,
p. 248).
The single-copy DNA to be used as the "tracer" is labeled
with radioactive Iodine-125. Single-copy DNA from a second species is used as the
"driver", and is not labeled. When the DNAs are mixed in the proportion of 1000
parts "driver" to one part "tracer", each "tracer" fragment
will reassociate with a "driver" fragment, forming a hybrid DNA fragment.Measuring the DNA distance. The two strands of a DNA
duplex are held together by hydrogen bonding, which depends on correct matching of base
pairs. The greater the extent of matching, the higher the temperature required to separate
("melt") them. The DNA distance is a measure of the reduction in melting
temperature of hybrid DNA fragments, caused by differences in their base sequences, and is
presumably a measure of the difference between the DNAs of the two species.
To determine the melting temperature of the hybrid fragments, they are
first bound to a hydroxyapatite column. The temperature is then raised in increments,
typically of 2.5ºC, and the column is washed, removing any DNA which may have separated
into single strands. The amount of DNA removed is measured by the level of radioactivity
in the sample (due to the Iodine-125 labeling). The percentage of DNA removed at each
temperature increment is plotted against temperature, producing a melting-point curve. In
the most common procedure, the temperature is recorded when 50% of the tracer DNA is
recovered in single-stranded form. This temperature is subtracted from the temperature at
which 50% of the pure "tracer" DNA melts. The result is called the "DT50H" (delta T50H), and is used as a measure of DNA distance.
In an alternative method, the measurements used are for only the tracer DNA fragments that
form duplexes with driver DNA. This result is called the DTm
(Sibley and Ahlquist 1983a, p. 257). The latter method should be used only when the
proportion of tracer DNA forming hybrid duplexes is greater than 80% (Sheldon 1987a).Normalized percentage of hybridization. It seems that
each species has some unique sequences, so that there is never a 100% match of the DNA
fragments of two species. The normalized percentage of hybridization (NPH) is the amount
of a species' DNA that hybridizes with that of another species, standardized against the
amount that hybridizes with DNA of its own species. High variances have been reported for
the NPH values for comparisons of closely related species (Bledsoe 1987), which would make
this measurement difficult to use in systematics. Under the experimental conditions used,
the NPH is often less than 75%. Because the non-hybridizing DNA will not attach to the
hydroxyapatite column, it is eliminated from the determination of the melting curve. If
the NPH is small, only a small fraction of the DNA remains for study, making the results
questionable.Delta T50H and DNA distance. Experiments to determine
the relationship between difference in DNA sequence and melting point change of DNA
duplexes have shown that a change of 1ºC in the melting point represents from 0.7% to
3.2% difference in DNA sequence, the best average estimates ranging from 1% (Bonner et al.
1973) to 1.6% (McCarthy and Farquhar 1972). Usually a DT50H
value of 1ºC is taken to indicate a difference of 1% in DNA sequences. Since the DT50H value and the percentage sequence difference are numerically
the same, I will use the term "DNA distance" for DT50H
values.

DISCUSSION OF RESULTS

Among the numerous papers published on the topic, two groups have
been selected for discussion. The study of the large flightless birds (ratites) was used
to calibrate the "DNA clock", and the studies of songbirds illustrate the
complexity of the results.Phylogeny of the ratites. The ratites are a group of
large flightless birds, including the ostrich, rheas, emu and cassowaries The kiwi and
extinct moas are also usually included, and the extinct elephant birds and some other
extinct groups are sometimes included as well (Cracraft 1974). The South American tinamous
are considered to be the closest relatives of the ratites. Because all the living and
recently extinct ratites are found on continental fragments of Gondwanaland (see
Figure 1) and share certain skeletal features, it has been postulated that they form
a natural group with a common ancestor which dispersed before the breakup of Gondwanaland.

The separation of Africa and South America due to the breakup of
Gondwanaland is believed to have occurred by the Late Cretaceous, about 80 million years
ago (Ma), according to conventional geological dating. This date has been used as an
estimate of the time of divergence of the ostrich and rhea. These two species show a DT50H of about 17.4 (Sibley and Ahlquist 1985d; first calculated as
15.7, Sibley and Ahlquist 1981), indicating about a 17.4% divergence of their respective
DNAs (Bonner et al. 1973). This figure was used to calibrate the "DNA clock" at
about 1% divergence per 4.6 Ma.
The DT50H values and calculated times of
divergence of the living ratites are shown in Figure 2. Since the "DNA
clock" is no longer considered reliable (Catzeflis et al. 1987), the divergence times
should no longer be defended. It appears that most of the ratites have extensive
differences in their DNAs, and may not be related. Fossil evidence (Houde and Olson 1981,
Olson 1985) has proposed that the ratites are not all related.

FIGURE 2. DNA-distance tree for the living ratites. To determine the DNA distance
between any two species, locate the point where the lines from the two species connect,
and read the DNA distance from the scale on the left.

Phylogeny of the songbirds. The order of perching
birds (Passeriformes) contains more than half the known species of birds (Bock and Farrand
1980). The New World flycatchers, ovenbirds and antbirds, and the Old World pittas and
broadbills are grouped together in one or more suborders known collectively as the
suboscines. The rest of the passeriforms are grouped in the suborder Passeres, or
songbirds. Three main divisions of songbirds are generally recognized (Storer 1971): the
corvine assemblage (crows, bowerbirds, birds of paradise, etc.), the predominantly Old
World ten-primaried group (thrushes, babblers, Old World warblers and flycatchers, wrens,
thrashers, etc.), and the predominantly New World "nine-primaried" assemblage
(finches, woodwarblers, tanagers, blackbirds, etc.).
Sibley and Ahlquist (1985a,b,c,d and references therein) have applied
the DNA/DNA hybridization technique to species representing most of the passeriform
families. Based on their results, they have proposed a classification involving numerous
taxonomic changes, many of which are significant departures from more traditional
classifications. They recognize (Sibley and Ahlquist 1985c) two main lineages of
songbirds: a crow-like assemblage, which includes a majority of the endemic Australian
species, and a second assemblage which includes the thrushes, sparrows, warblers and most
other species from non-Australian groups.
The taxonomic changes proposed by Sibley and Ahlquist involve
considerable re-grouping of genera, splitting certain families and joining the fragments
to various other families and erection of new families from pieces of old families. The
extent of the proposed changes can be illustrated by comparing the composition of their
family Corvidae with the more traditional classification (see Table 1). Quite
naturally, there has been a certain amount of resistance to some of these suggestions. It
should, however, be pointed out that several of the genera of birds involved in the
controversy have been rather puzzling taxonomically. The DNA-based classification at least
provides a new approach to the problem of classifying them. Several of the challenges to
the method are discussed later in this paper.

TABLE 1. Comparison of classification of members of Corvidae according to Sibley and
Ahlquist (1985a) with their classification in Bock and Farrand (1980).

Common Name

Old Classification

New Classification
(Family Corvidae)

cuckoo-shrikes

Family Campephagidae

Subfamily Corvinae

wood shrike

Family Laniidae
Subfamily Pityriasinae

Subfamily Corvinae

whipbird

Family Muscicapidae
Subfamily Orthonynchinae

Subfamily Cinclosomatinae

silktail

Family Muscicapidae
Subfamily Sylviinae

Subfamily Monarchinae

Peltops flycatcher

Family Muscicapidae
Subfamily Muscicapinae

Subfamily Corvinae

monarch flycatchers

Family Muscicapidae
Subfamily Monarchinae

Subfamily Monarchinae

fantails

Family Muscicapidae
Subfamily Rhipidurinae

Subfamily Monarchinae

whistlers

Family Muscicapidae
Subfamily Pachycephalinae

Subfamily Pachycephalinae

Australian nuthatches

Family Sittidae
Subfamily Daphoenosittinae

Subfamily Pachycephalinae

figbird

Family Oriolidae

Subfamily Corvinae

drongos

Family Dicruridae

Subfamily Monarchinae

magpie-lark

Family Grallinidae
Subfamily Grallininae

Subfamily Monarchinae

apostlebird

Family Grallinidae
Subfamily Corcoracinae

Subfamily Corcoracinae

woodswallows

Family Artamidae

Subfamily Corvinae

currawongs

Family Cracticidae

Subfamily Corvinae

birds of paradise

Family Paradiseaeidae

Subfamily Corvinae

crows

Family Corvidae

Subfamily Corvinae

A diagram of a portion of the classification proposed by Sibley and
Ahlquist (1985c) is shown in Figure 3. Note that the DNA distance between branching
points is often very small. In such cases the order of branching is highly uncertain, even
if the branches are statistically distinguishable. Note also that groups of species tend
to form distinct clusters at lesser DNA distances, the clusters joining together in a
series of closely spaced nodes at DNA distances of about 8 or greater. Some possible
interpretations of these tendencies will be discussed later.

FIGURE 3. Partial phylogeny of songbirds. Based on DNA-DNA hybridization. To
determine the DNA distance between two species, find the horizontal line joining the lines
from the two species and read the DNA distance from the scale on the left of the diagram.
After Sibley and Ahlquist 1985c, Fig. 16.

In considering the usefulness of DNA distances in classification,
two aspects of systematics can be considered separately. One goal of systematics is to
identify the group to which a particular species belongs. Another goal is to estimate the
degree of relatedness of various species groups. Examples have been chosen from the
literature to illustrate the use of DNA distances in attempting to satisfy each of these
goals. The success of the technique will be evaluated by its ability to produce distinct
clusters of species. Different clusters should be separated by gaps that are greater than
the range of values for species within a cluster.
DNA distances for comparisons of three species of thrashers (Family
Mimidae) to several species of songbirds are shown in Figure 4. Note the small
distances among the thrashers, and their isolation from species in other families. One
species, Donacobius atricapillus (see arrow in Figure 4), is classified with
the thrashers in Peter's checklist (Mayr and Greenway 1960), but is far from the thrashers
using DNA distances (Sibley and Ahlquist 1984b). The latter authors suggest it may be a
wren, but have not measured the DNA distance involved. If Donacobius is truly a
wren the usefulness of the DNA/DNA hybridization technique in classification would be
supported.

FIGURE 4. DNA distances to several species of songbirds, for three species of
thrashers, Toxostoma longirostre (·), Mimus
polyglottus (*), and Dumetella carolinensis (o). The first four genera
listed are thrashers (family Mimidae); the next ten are starlings (Family Sturnidae). Note
that the DNA distance from any of the thrashers to any of the starlings falls in the range
of 6.2 to 6.5. Note also the lack of values (gaps) between DNA distances of 2.8 to 5.2 and
from 6.5 to 8.2. Data from Sibley and Ahlquist 1984b and Sibley, Ahlquist and Sheldon
1987.

Referring again to Figure 4, note that the starlings are
grouped together, and are separated from the thrashers at DNA distances ranging from 5.0
to 6.4. Note also that the range of values within the starlings (1.4) is less than the gap
(2.3) separating the family from the next closest group (Turdinae + Muscicapinae). This
result suggests the surprising possibility that starlings may be the closest relatives of
mockingbirds. If so, the DNA/DNA hybridization technique has been useful in the second
goal of systematics mentioned above, determining which other groups are most similar to a
given group of species. However, gaps between groups become less than ranges within groups
for DNA distances above 8, and DNA distances between different groups run together at
values over 10. This pattern of distinct clusters at values less than about 8, and joining
of many clusters at values over about 8 or 10 is also seen in comparisons for several
other genera (Sibley and Ahlquist 1982a,b; 1985d), and suggests that DNA distances are
most meaningful for values less than perhaps about 8.
A somewhat different situation is illustrated by the DNA distances for
Australian treecreepers, shown in Figure 5. Two species of treecreepers are separated
by a DNA distance of 5.4, an unusually high figure for species in the same genus, leading
to the suggestion they be placed in separate genera (Sibley, Shodde and Ahlquist 1984). No
other family seems close to the treecreepers based on DNA distances. The closest species
are bowerbirds, but the DNA distances (>10) are not sufficiently distributed to clearly
indicate relationships. Thus it appears that either the treecreepers have no close
relatives, or their relatives were not included in the tests, or the tests were
unsuccessful in identifying them.

FIGURE 5. Some DNA distances for two species of Australian treecreepers, Climacteris
rufa (*) and C. picumnus (·), Family
Climacteridae. Note the unusually large DNA distance of 5.4 between the two species of Climacteris,
and the large gap separating the genus Climacteris from all other genera. Data
from Sibley, Schodde and Ahlquist 1984.

Not all DNA distance experiments produce distinct clusters of
species. DNA distances for the blue vanga of Madagascar are shown in Figure 6. No
distinct clusters are seen, nor any very close relatives. A similar lack of meaningful
clustering is shown by the Australian "nuthatches" (Sibley and Ahlquist 1982c).
Another problematic result is illustrated in Figure 7. Here the DNA distances for the
olive bush-shrike are shown. Note the lack of distinct clustering among the values. The
gaps that do exist are small, and do not appear to have biological significance. In these
instances, the DNA/DNA hybridization method does not seem to have given a clear answer to
the question of the relationships of the species involved.

FIGURE 6. DNA distances to several species of songbirds, for the blue vanga, Leptopterus
madagascarinus, Family Vangidae. Note the lack of significant gaps in the range of
values. Data from Sibley and Ahlquist 1985d.

FIGURE 7. DNA distances to several species of songbirds, for the olive bush-shrike, Telophorus
olivaceous, Family Laniidae. Note the lack of significant gaps in the distribution of
the values. Data from Sibley and Ahlquist 1985d.

CRITICISMS OF DNA HYBRIDIZATION METHODOLOGY

Several papers critical of various aspects of DNA hybridization have
recently been published (Templeton 1985, 1986; Britten 1986; Houde 1986, 1987; Cracraft
1987). The criticisms may be divided into those pertaining to the assumptions, the
experimental technique and the interpretation of the data (Houde 1987). I will discuss
several of the criticisms that have been raised.Criticisms of the assumptions of the method.
Acceptance of the method of DNA hybridization is based on the acceptance of certain
assumptions, such as that all single-copy DNA sequences have homologs with which they can
hybridize, and all degrees of divergence can be detected (Sibley and Ahlquist 1983a, p.
257). Another assumption is that nonhomologous sequences will not be similar enough to
hybridize (Sibley, Ahlquist and Sheldon 1987, p. 114). However, each of these assumptions
is sometimes violated (see Templeton 1986, Zweibel et al. 1982).
The first assumption is violated because different lineages probably
have unique sequences, without homologs in other lineages. If homologous sequences have
diverged beyond the 75-80% matching required for duplex formation (Sibley, Ahlquist and
Sheldon 1987), homology could not be detected by the method.
The second assumption is violated because not all degrees of divergence
can be detected. For example, random reassociation is reported below about 45ºC (Sibley
and Ahlquist 1981, p. 305), making analysis impossible below this temperature. To avoid
this problem, the beginning temperature used experimentally is usually about 60ºC. Since
most homologous hybrids melt at about 80-85ºC, the method can be applied only for DT50H values less than about 20-25 units. Even this range may be too
great, as a rapid departure from a linear relationship is reported at DT50H
greater than about 15 (Houde 1987; Sibley and Ahlquist 1985d, p. 152).
The assumption that nonhomologous sequences will not form duplexes may
be true in most cases, but it appears that some nonhomologous proteins do have similar
amino-acid sequences (see Hill and Hastie 1987, Schwabe and Warr 1984), implying similar
DNA sequences and the possibility of nonhomologous matching in duplex formation.Criticisms of experimental methodology. The
experimental methodology has been criticized on two main counts: experimental uncertainty
and the nature of the DNA sequences being compared.
Variation in fragment size may cause inconsistencies in experimental
results (Sibley and Ahlquist 1981, p. 307; Sibley, Ahlquist and Sheldon 1987, p. 104).
Differences in fragment length can alter the melting temperature by 1.5 to 2.5ºC (Caccone
and Sbordoni 1987). Variation between individuals of a species can also affect
experimental error (Cracraft 1987), and should be better analyzed. However, in
hybridizations involving six species and subspecies of juncos, the maximum DTm distance detected was only 0.2 (Shields and Straus 1975). The
range of DT50H values among 13 individuals of the American
robin was 2.1 (Sibley and Ahlquist 1983a). Repeated measurements using the same material
may vary by 0.6ºC (Sibley and Ahlquist 1983a, p. 265). Overall uncertainty has been
estimated as from 0.4 (Shields and Straus 1975) to about 2 (Sibley and Ahlquist 1981, p.
319) or possibly even as high as 3.1 (Sibley and Ahlquist 1983a, p. 275). It appears that
the total uncertainty in comparing two DNA distances based on single measurements could be
large enough to cause problems in interpretation.
Another question concerns the nature of the DNA used in the
experiments. The distinction between single-copy and multiple-copy DNA depends on
experimental conditions (McCarthy and Farquhar 1972). If DNA from different species reacts
in different ways, it seems possible that the DNA samples from the two species might not
be equivalent qualitatively or quantitatively. How well the technique separates the
single-copy DNA is not clear (see Sibley and Ahlquist 1981, p. 319), nor is it certain
that the single-copy DNA contains the sequences that distinguish species. Species
differences may be determined by developmental control sequences, which may be found among
the moderately repetitive sequences. The sometimes considerable differences between
phylogenies based on DNA distances and those based on morphology raise the question of
whether the appropriate data are being collected. (For further discussion of this point,
see below under section titled "DNA distance and relationships".)Criticisms of data interpretation. The most severe
criticisms of the DNA distance method have been directed against the method of tree
construction and the accuracy of the "DNA clock".
The validity of using a single distance figure to construct
phylogenetic trees is open to question. DNA distance may not be an accurate indicator of
branching order if it is not well correlated with time. This is because the DNA distance
is the sum of the differences between two species. Species that diverge slowly might have
smaller DNA distances than species that diverged rapidly, regardless of time since
divergence. Simply showing that two measurements are statistically different (e.g., Sibley
and Ahlquist 1987) does not necessarily indicate their order of branching. Since it has
been shown that rate differences exist (Catzeflis et al. 1987, Houde 1987, Sheldon 1987b),
trees based solely on DNA distance should be checked against other methods.
Another problem with the method of tree construction is that branching
points within a tree are often separated by less distance than the experimental
uncertainty (usually estimated at about 1.0, but possibly up to 3.0 units, Sibley and
Ahlquist 1983a, p. 275). Branches of a tree should be separated by distances greater than
the experimental uncertainty if the data are to be used to determine the order of
branching.
Another criticism of the method of tree construction is that it is not
cladistic, that is, the DNA hybridization method makes no distinction between "shared
derived" sequences and "shared primitive" sequences. Instead, it is simply
an attempt to sum the unique (autoapomorphous) sequences of two species. In addition,
Houde (1987) has pointed out that the branching pattern of a DNA distance tree depends on
the clustering method used, and Lanyon (1985) has shown that omission of a single taxon
can affect the branching order of the remaining taxa. This lack of stability of some DNA
trees under differing conditions weakens confidence in the method.
Phylogenetic trees based on DNA distance often conflict with trees
based on other types of data (e.g., Sibley and Ahlquist 1984a, 1985a,b,d; Lanyon 1985).
Agreement of DNA distance data with morphological data is good when species are
morphologically similar, but declines as morphological differences increase, such as at
the levels of family and order (Sibley and Ahlquist 1983a, p. 278). Three phylogenies of
the superfamily Tyrannoidea, each based on a different database, were compared by Lanyon
(1985). The three databases were based respectively on morphology, protein
electrophoresis, and DNA/DNA hybridization (see Figure 8). Each phylogeny was
different, with no agreement of all three methods on any relationships at the family or
subfamily levels. Lanyon (1985) suggested that the three groups may all have arisen at
essentially the same time. The lack of agreement between different methods may suggest the
existence of separate lineages. On the other hand, it might be that the three groups are
descended from a common ancestor with a high degree of genetic variability, or that point
mutations are not the only factor affecting measurements of DNA distance.

FIGURE 8. Comparison of trees for four species of suboscines. Based on three
different data sets M = Myiarchus, S = Schiffornis,
T = Tityra, P = Pipra.

Some comments on the DNA clock. Although Sibley and
Ahlquist have backed away from their earlier insistence that their method is clock-like
(Catzeflis et al. 1987), the idea is still considered useful (Sibley and Ahlquist 1987)
and a few comments concerning the "clock" are given below.
The theoretical basis for the DNA distance clock rests on the
assumption of the importance of neutral mutations. Yet the method is designed to eliminate
most of the DNA in which mutations seem most likely to be neutral, the repetitive DNA.
Mutations in translated sequences of the single-copy DNA may be subject to natural
selection to a significant extent. If true, different branches of a lineage might diverge
at different rates, making it impossible to reconstruct the order of branching from the
distance data. The importance of this effect would depend on the relative proportions of
translated DNA and non-translated intervening sequences.
Mutation rate differences of 25-50% have been reported among herons
(Sheldon 1987b) and primates (Fitch 1986; Bonner, Heinemann and Todaro 1980). Catzeflis et
al. (1987) estimated the rate for rodents to be ten times the rate for hominoid primates.
These estimates are based on evolutionary assumptions concerning the time since divergence
from a hypothetical common ancestor, and show that the proposed clock is not consistent
with the evolutionary assumptions.
It is difficult to compare estimates of time since divergence based on
DNA distances, with estimates based on the fossil record. Fossil songbirds are not
abundant and are difficult to identify even to family. However, no fossil songbirds are
known before the Miocene (Brodkorb 1987, Olson 1985), while the proposed DNA distance
clock suggests divergence of most families by the end of the Eocene (Sibley and Ahlquist
1985c). This difference has not been resolved satisfactorily.
The reason for differences in mutation rates is not yet known. It has
been suggested (Britten 1986; Li, Tanimura and Sharp 1987) that mutation rate may depend
on the number of DNA replications per year. This hypothesis has not been adequately
studied, but does not appear to be satisfactory. Another suggestion (Britten 1986) is that
differences in mutation rates are due to differences in efficiency of DNA replication and
repair enzymes. Another possibility that deserves more attention is that many differences
in DNA sequences maybe the result of the original creation of separate lineages rather
than to divergence from a common ancestor.

WHAT DOES DNA DISTANCE MEAN?

Despite the shortcomings of the DNA/DNA hybridization method, there
appear to be some interesting patterns in the results. It seems useful to attempt to
evaluate the data to determine whether it can be meaningfully interpreted within a
creationist philosophical framework.Causes of DNA sequence similarities. Similarities in
organisms could come about from a number of different causes (see Coyne and Barton 1988,
Gibson 1986). Neither chance nor convergence seem plausible as causes of DNA sequence
similarity. Sibley and Ahlquist (1985c, p. 84) state that 80% homology is required to form
a stable DNA duplex at 60ºC. Random changes do not seem likely to create 80% similarity
in a sequence of 500 base pairs, even if aided by selection. The possible importance of
constraints on genetic variation as a cause of convergence is not known. Cross-species
gene exchange has been suggested as a cause of DNA similarity (Syvanen 1987), but it is
not well understood, and seems to be uncommon.
There are at least two other possibilities for explaining similarities
in DNA sequence: common ancestry and common design. Creationists accept both factors as
valid. The question of interest here is whether the DNA distance data show any pattern
that might be useful to distinguish common ancestry from common design. The following
sections pursue this question further.How rapidly do DNAs diverge? The rate of change of DNA
sequences has never been measured directly, so it is difficult to estimate how much DNA
divergence is plausible within 5,000 to 10,000 years. No practical method is available
that detects all point mutations in an entire genome, although it is possible to clone and
sequence a gene and compare genes from different species. To be meaningful, variation
within a population would have to be distinguished from variation between populations. It
is problematic whether the results could be extrapolated to entire genomes.
Experimentally detected spontaneous mutations appear to be rare. Most
estimates of mutation rate are based on protein electrophoresis, and are minimum rates
because electrophoresis detects only mutations resulting in a substitution of an amino
acid having different charge characteristics. Overall mutation rates are greater by an
unknown amount. Mutation rates for laboratory mice have been estimated at about 10-6
per locus (gene) per generation (Neel, Mohrenweiser and Mesiler 1980; Russell et al. 1979;
Johnson and Lewis 1981; Johnson et al. 1985). This is about 10-9 per nucleotide
per generation (assuming an average of 103 amino acids per locus, see
Table 2 in Jukes 1980). A mutation rate of 10-8 (per nucleotide) as an
average for the entire genome would suggest a rate of DNA divergence on the order of 1%
per Ma, or 0.01% per 10,000 years.
The highest mutation rate known for a human genetic disease (Duchenne
muscular dystrophy) is about 10-4 (Rotter and Diamond 1987, Moser 1984). This
seems a very high rate for a deleterious mutation, and suggests that there is much more to
learn about mutation rates, such as the causes of mutational "hot spots". The
DNA/DNA hybridization technique is probably sensitive to major differences in DNA
sequence, but may not be able to distinguish small differences from experimental
uncertainty. The questionable ability of the method to identify homologous sequences also
weakens any confidence one might like to have in any estimate of mutation rate based on
DNA distance. Since DNA distance is not considered to be linear with time (Sibley and
Ahlquist 1985c, Catzeflis et al. 1987), estimates of mutation rates based on DNA distance
might not be meaningful. Rough estimates are from about 0.1% per Ma to more than 1% per Ma
(Britten 1986, Table 1). These estimates are based on assumptions of a hypothetical common
ancestor and a datable speciation event.
Determinations of DNA distance between populations of known historical
age are not available. Many historically dated introductions are known, and measurements
of DNA distances among them would be of interest. If different individuals of a species
may differ by more than 2 DNA distance units (Sibley and Ahlquist 1983a), it seems likely
that newly formed daughter species could differ by that amount at the time of their
formation.
Based on a study by Fitch and Atchley (1985), Lewin (1985) suggested
that mutation rates in inbred laboratory mice may be as high as about 5×10-4
per locus, which would be about 5×10-7 per nucleotide pair per generation. The
rate of DNA divergence calculated from this mutation rate would be on the order of 0.5%
divergence per 10,000 years, far higher than previous estimates. However, other
explanations of the data have been offered (Bishop et al. 1985, Bonhomme et al. 1987,
Green et al. 1985, Johnson et al. 1985). The present interpretation (Fitch and Atchley
1987) seems to be that the original breeding stock came from a cross between two
subspecies and that a high degree of heterozygosity has been maintained in the breeding
stock. Thus there seems to be no presently accepted evidence that inbred mice have high
mutation rates.DNA distance and relationships. It seems likely that
all species in a genus of birds share a common ancestry. DNA distances for congeneric
birds are usually less than about 3.0, although distances as high as 5.3 have been
recorded (Sibley and Ahlquist 1985d). Experimental uncertainty can easily explain
distances of 2.5 (see above under criticisms of methodology). Distances greater than 4 or
5 are not so easily explained by experimental uncertainty, and another explanation should
be sought.
Differences in DNA among genetically related species may not be due
exclusively to point mutations. Viruses, movable elements, and chromosomal deletions and
rearrangements may affect comparisons of DNA sequences. The effects of these factors
should be investigated. Natural selection may also affect DNA sequences greatly enough to
affect DNA distances. If most mutations are subject to natural selection rather than being
neutral, it will be necessary to reevaluate the use of DNA sequence comparisons in
estimating mutation rates and in estimating the importance of neutral mutations in
evolution.
On the other hand, species may have been created with a high degree of
genetic variability, with many different genes acting on single traits (polygenes) and
many alternative forms of genes (multiple alleles). Speciation could then result in
division of the original gene pool with concomitant differences in DNA sequences without
the need for mutations (see Lester and Bohlin 1984, p. 168). The result would be
increasing specialization and loss of adaptability, trends well illustrated in insular
faunas.
The fact that DNA distance is sometimes poorly correlated with other
methods of classification illustrates the difficulties in classification. The example of
the groups of New World tyrant flycatchers has already been mentioned. A few other
examples are described in the paragraphs below.
The barbets (family Capitonidae) are believed to be related to the
woodpeckers, and are found in both Africa and South America. DNA distances between species
of this family are as high as 17.4 (Sibley and Ahlquist 1985d). The explanation given is
that the African and South American groups are only distantly related, although they are
morphologically similar. On the other hand, the DNA distance between pelicans and New
World vultures, classified in different orders, is only 9.7 (Sibley and Ahlquist 1985d).
This latter figure is about the same as the DNA distance between kinglets and Old World
warblers, both from the same subfamily (Sibley and Ahlquist 1985c).
Species from different subfamilies of vireos may have DNA distances of
only 4.1 (Sibley and Ahlquist 1982b). Compare this with the DNA distance of 5.3 for two
species in the same genus of treecreepers (Sibley, Schodde and Ahlquist 1984). One would
expect species from the same genus to have smaller DNA distances than species from
different subfamilies. It appears that similar DNA distance values may not have the same
significance in different groups, and may not always be accurate indicators of
relationships.
Although taxonomic groupings determined by DNA distances are often
congruent with those determined by morphology, the number of inconsistencies is
substantial. Unless some independent evidence can be found to support the relationships
proposed from DNA distances, it seems reasonable to suggest that a point is reached beyond
which the DNA/DNA hybridization method is not useful in determining relationships. On the
other hand, if morphology is truly subject to convergence as much as is suggested by the
DNA/DNA hybridization technique, classifications based on morphology need to be
reevaluated. The usefulness of fossils in tracing ancestry would also be seriously
challenged, since morphology is the basis for comparing fossils.Design vs. ancestry. One goal of creationism would be
to search for some method of distinguishing differences in design from differences
resulting from divergence from a common ancestor. It would be desirable to find a method
that clusters species into separate and distinct groups. The DNA distance method seems to
be able to do this for some groups of songbirds (see Figure 4) but does not do so
well with others (see Figures 6,7).
Since DNA distances for songbirds seem to produce species clusters at
low values, and these clusters often seem distinct at DNA distances of about 8 or less
(see Figure 3), one could propose that the species within such a cluster may be truly
related by ancestry. Distances greater than 8 or 10 appear to be of limited usefulness in
determining relationships, either because of limitations of the technique, or because such
differences are due to separate ancestries.
Additional data would be helpful in testing whether these figures are
plausible. Especially interesting would be determination of DNA distances for the
following: populations of known historical age, such as various breeds of pigeons or other
domesticated species; similar species with greatly differing chromosomal banding patterns,
such as the muntjacs; and groups with disjunct distributions, such as parrots or trogons.
Complete matrices of DNA distances for all species in a few related groups would also be
of interest. Such information could significantly affect the above tentative
interpretations.

SUMMARY AND CONCLUSIONS

DNA strands are held together by hydrogen bonding between
complementary bases in the DNA. The closer the matching of the two strands, the higher the
temperature required to separate them. DNA distance is the number of degrees Celsius the
melting temperature of a hybrid DNA duplex is lowered because of mismatching of DNA bases
from the two different species. DNA distance is used as a measure of the similarities in
base sequence of DNA from two species, and is used as an estimate of the closeness of
their relationship. However, as the difference between two species increases, the amount
of DNA able to form duplexes decreases. If the portion forming duplexes is less than
perhaps 90-95% of the DNA fragments, the results may be of questionable significance.
Phylogenies based on DNA/DNA hybridization have been constructed for
several groups, including ratites, herons and songbirds. Among songbirds, the following
trends are noted:

DNA distance values using a single tracer species are typically not continuously
distributed, but show gaps at values less than about 8. At values above about 10, the
values become more nearly continuously distributed (Figure 4).

Species often tend to form clusters that are separated from other clusters at DNA
distances of about 6 to 9 (Figure 3).

Nodes joining species clusters at distances of about 10 or more are so close together as
to make sequencing of the nodes highly questionable (Figure 3).

DNA distances seem to be useful in assigning a species to a group for values less than
about 6 or 8. Occasionally, a DNA distance value is unexpectedly high, suggesting a
species has been misclassified (e.g., Donacobius, Figure 4).

DNA distances sometimes indicate a probable genetic similarity of two groups of species
(e.g., mimids and sturnids, Figure 4) at values less than about 8.

DNA distances of the same magnitude do not necessarily have the same significance in
different groups (see above discussion under DNA distance and relationship).

These trends suggest the following tentative conclusions:

Low DNA distances (less than 5 or 6) between species of songbirds suggest genetic
similarity, and probable common ancestry if supported by other data.

High DNA distances (above 10) suggest genetic differences, but are not necessarily
indicative of evolutionary relationships between groups. Instead, they may suggest
different ancestries.

The results of DNA distance experiments are sometimes unclear and should not be used as
the sole basis for a phylogeny.

Equal DNA distances in different groups do not necessarily have equivalent meaning.
Results from one group should not be extrapolated to another group.

To this creationist, the DNA-distance data present interesting
hypotheses that might not have been thought of without the technique. From this view, the
method is interesting and stimulating. However, the method is fraught with difficulties
similar to those of other methods of systematics. The ability to cluster species into
groups is interesting and may be useful. Attempting to infer time since divergence or the
precise order of speciation events seems to require too much from the method. More data
would be of interest, especially complete matrices for family groups, and DNA distances
between isolated populations of known historical age. Such data will continue to be of
interest to those interested in developing a modern creationary theory of systematics.

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